FEBS Letters 581 (2007) 1217–1226

STAT1 regulates p73-mediated Bax gene expression

Surinder M. Soonda,*, Christopher Carrolla, Paul A. Townsendb,1, Emre Sayanc,2, Gerry Melinoc,2,Iris Behrmannd,3, Richard A. Knighta, David S. Latchmana, Anastasis Stephanoua

a Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UKb Human Genetics Division, University of Southampton, Duthie Building, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, UK

c MRC Toxicology Unit, Hodgkin Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, UKd Laboratoire de Biologie et Physiologie Integree, Fac. Sciences, Techno. & Com., Universite du Luxembourg,

162A, avenue de la Faıencerie, L-1511, Luxembourg

Received 5 January 2007; revised 16 February 2007; accepted 22 February 2007

Available online 1 March 2007

Edited by Varda Rotter

Abstract Although signal transducer and activator of transcrip-tion 1 (STAT1) mediated regulation of p53 transcription andapoptosis has been previously reported, modulation of othermembers of the p53 family of transcription factors remainspoorly understood. In this study, we found that STAT1 andTA-p73 can interact directly and that p73-mediated Bax pro-moter activity was observed to be reduced by STAT1 expressionin a p53-independent manner for which STAT1 Tyrosine-701and Serine-727 are key residues. This study presents the firstreport physically linking STAT1 and TA-p73 signalling andhighlights the modulation of the Bax promoter in the contextof IFN-c stimulation.� 2007 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.

Keywords: Signal transducer and activator of transcription 1;Interferon-gamma; TA-p73; p53; Bax

1. Introduction

Originally identified as key intermediates in interferon (IFN)

signalling [1], the STAT (signal transducer and activators of

transcription) proteins have gained considerable attention re-

cently as regulators of cell growth and apoptosis [2,3]. In order

to activate STAT1 fully, phosphorylation of Tyrosine-701 by

Janus kinase (JAK) and dimerisation has been shown to be a

pre-requisite in addition to the phosphorylation of STAT1

on Ser-727 in a mitogen-activated protein kinase-dependent

manner [4–6]. As a key transcription factor activated during

stress-response signaling, STAT1 has also been demonstrated

to interact with p53 and potentiate apoptotic signaling [7]. Tar-

get genes that have been demonstrated to be transcriptionally

enhanced by the combination of STAT1 and p53 activation in-

Abbreviations: STAT1, signal transducer and activator of transcription1; MEF, murine embryonic fibroblast; IFN-c, interferon-gamma;GST, glutathione S-transferase; PBS, phosphate-buffered saline; aa,amino acids

*Corresponding author. Fax: +44 0 207 905 2301.E-mail address: s.soond@ucl.ac.uk (S.M. Soond).

1Fax: +44 23 8079 4264.2Fax: +44 116 252 5616.3Fax: +352 466 644 435.

0014-5793/$32.00 � 2007 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2007.02.049

clude p21waf [8,9], Bcl-2 associated protein X (Bax), Noxa and

Fas [7].

As member of the p53 transcription factor family, TA-p73 is

a modular protein whose activity is under stringent control, in

a transcriptional, translational and post-translational manner

[10]. The TA-p73 protein is expressed as 7 functionally distinct

C-terminal spliced variants arising from the alternative splicing

of the TA-p73 mRNA [11–13]. Trans-activating TA-p73 can

bind p53-responsive elements and activate p53 target genes in-

volved in cell cycle arrest and apoptosis [14].

The Bcl-2 family of anti-apoptotic and pro-apoptotic pro-

teins, all of which possess one or more Bcl-2 homology

domains, are regulated by post-translational modification,

sub-cellular localization and dimerisation [15]. As a pro-apop-

totic member of this family, Bax is transcriptionally up-regu-

lated by TA-p73 (as is PUMA). The time kinetics of this

event during the induction of cell death is consistent with

PUMA mediated Bax translocation, rather than expressed-

TA-p73 directed Bax translocation [16], highlighting a pro-

apoptotic role for TA-p73.

Considering that the Bax promoter can also be positively

regulated by p53 [7,17], the underlying mechanisms that allow

the selective TA-p73 mediated Bax regulation in the absence of

p53 mediated Bax expression, remain uncharacterized. In this

context, key regulators and up-stream signaling intermediates

of TA-p73 and p53 have received considerable attention. As

we will show in this study, one such protein that is common

to the regulation of p73 (as well as p53) mediated gene regula-

tion is STAT1.

We show that STAT1 can physically associate with TA-p73

in vitro and in vivo, that TA-p73-directed Bax promoter activ-

ity and protein expression is significantly reduced by STAT1

expression in mouse embryonic fibroblast (MEF) cells in the

presence and absence of IFN-c stimulation and that this can

occur in a p53-independent manner.

2. Materials and methods

2.1. MaterialsMouse and rat IFN-c were purchased from Sigma (UK). Rabbit

anti-STAT1 (sc-346), mouse anti-phospho 701 STAT1 (for use in co-immunoprecipitations, Sc-8394), rabbit anti-Bax (sc-526), rabbitanti-HA probe antibody (Y-11) and anti-b-actin were purchased fromSanta Cruz Biotechnology. Mouse anti-phospho-701 STAT1 (Zymed)was used for Western blotting alone. Transfections were performedusing Fugene 6 (Roche) and used as recommended. The human

blished by Elsevier B.V. All rights reserved.

1218 S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226

Bax-luciferase reporter plasmid was a generous gift from G. Melino(MRC Toxicology Unit, University of Leicester). Anti-Myc antibody(Invitrogen), anti-HA antibody (clone 3F10, Roche), GAP-DH(MAB 374, Chemicon) and mouse anti-STAT1 (SM2 mouse monoclo-nal, Abcam) were also used in this study. Normal IgG antibodies andProtein A/G-PLUS sepharose were purchased from Santa Cruz Bio-technology.

2.2. PlasmidsSTAT1-pGEX5X-2 was a gift from Behrmann [18]. Mammalian

Expression plasmids for HA-tagged human TA-p73 derived proteinsand bacterial expression plasmids for human TA-p73 derived glutathi-one S-transferase (GST)-fusion proteins were gifts from G. Melino(University Of Leicester) and have been described previously [12,19].Human STAT1-a/MYC-pCDNA6A, STAT1-b/pRc/CMV, STA-T1Y701F/pRc/CMV, STAT1S727A//pRc/CMV and mouse STAT3/pCDNA3 expression plasmids were a generous gift from J. Darnell(Rockefeller University, New York, USA) and have been describedpreviously [20]. The mammalian expression plasmid for HA-tagged hu-man p53 expression plasmid (p53-HA) was a gift from G. Melino (Uni-versity of Leicester).

2.3. Cell lines and reagentsCOS-7 cells were maintained in Dulbecco’s modified Eagle’s medium

(DMEM) as described previously [21]. STAT1 deficient MEF cells(STAT1�/� MEF) and their wild-type counterparts were maintainedin supplemented DMEM (as above) containing 10% v/v FBS and,non-essential amino acids and were a gift from David E. Levy [22].Mice deficient for STAT1 and p53 were generated by crossingSTAT1/129S6/SvEv [23] deficient mice (purchased from Taconic, Den-mark) with p53/C57BL/6J [24] deficient mice (Taconic) giving rise todouble heterozygous mice, which were cross-bred with each other giv-ing STAT1/p53�/� deficient embryos. Embryos were isolated at 13.5days PC and MEF cells harvested following trypsin digestion and cul-tured in DMEM containing 10% FBS with the above supplements andgenotyped for being STAT1/p53�/� ‘double-knockout’ by PCR usingprimers specific for STAT1 and p53 and by Western blotting.STAT1/p53�/� MEF cells were pooled and transfected with DNAusing Fugene 6 reagent after 3 passages.

2.4. Expression and purification of GST fusion proteinsGST expression plasmids were used to transform competent BL-21

LysS (Promega) cells, which were then grown to log-phase at 37 �C, in-duced with 1 mM IPTG for 3 h at 30 �C and GST fusion proteins iso-lated by immobilization on glutathione Sepharose (AmershamBiosciences) as outlined [21].

2.5. GST co-precipitation and co-immunoprecipitation assaysTransfected cells were lysed in 1% v/v Igepal-630, 50 mM Tris pH

8.0, 150 mM NaCl, 10% v/v glycerol, 5 mM EDTA, 1 mM PMSF,20 lg/ml leupeptin, 10 lg/ml aprotinin, 1 mM NaF, 1 mM Na3VO4

(1%NP-40LB) and the cleared lysates were incubated with approxi-mately 5 lg GST-Sepharose, GST-STAT1 or -p73 derivative-Sepharose, incubated overnight at 4 �C, washed 6 times in 0.1% v/vIgepal-630, 50 mM Tris pH 8.0, 150 mM NaCl, 10% v/v glycerol, 5mM EDTA, 1 mM PMSF, 20 lg/ml leupeptin, 10 lg/ml aprotinin, 1mM NaF, 1 mM Na3VO4 (0.1%NP-40LB) and analysed by Westernblotting as described [21]. Identical lysis conditions were used forco-immunoprecipitation of proteins and have been described [25].

2.6. Confocal microscopySTAT1�/� MEF cells were seeded on glass coverslips at a density

of 103 cells per 13 mm coverslip and transfected the following dayusing Fugene 6 and incubated for another 24 h. Cells were fixed inParaformaldehyde (PFA), permeablised in PFA, 0.1% v/v Triton X-100 and stained using antigen specific antibodies as outlined [25].Alexa-Fluor 488 donkey anti-mouse IgG, Texas Red goat anti-ratconjugates were used as secondary antibodies (10 lg/ml) and werepurchased from Molecular Probes (Invitrogen). Coverslips weremounted in Vecta Shield mounting mix containing DAPI (Vecta Lab-oratories, UK), visualized at room temperature using a Leica TCSSP2 Laser Scanning Confocal Microscope and with 63· objectivesand analysed using LCS Lite (Leica) and Adobe Photoshop 6.0software.

2.7. Luciferase reporter assaysMEF cells were seeded at 4 · 105 cells per well for 6 h, transfected (in

duplicate) using DNA/Fugene 6 complexes and the incubation contin-ued for 48 h at 37 �C.Human Bax-luciferase reporter plasmid was agift from G. Melino (University of Leicester). For standardization oftransfection efficiencies, 50 ng of b-galactosidase/pRSV plasmid wasalso added to the transfection mix. Following incubation and stimula-tion the cells were washed in 1· PBS (lacking Ca2+ and Mg2+ ions) andlysed in reporter assay lysis buffer. 5 ll of cleared lysate was assayedfor luciferase activity using the Luciferase Assay System (Promega)and for b-galactosidase activity using the Beta-Glo system (Promega).Luciferase data were recorded (in duplicate) using a TD-20e lumino-meter (Turner Designs, USA) and the data presented are from oneset of experiments for which consistency was observed for at least 3times.

2.8. Isolation of rat cardiomyocyte cellsTwo days old Sprague–Dawley neonatal rats were used as heart tis-

sue donors from which cardiomyocyte cells were isolated and main-tained as outlined previously [26].

2.9. Statistical analysisLuciferase data were analysed using Microsoft Excel (Microsoft,

USA) to determine the standard error around the mean, before the re-sults were translated to fold activation of relative light units over basalcontrol experiments.

3. Results

3.1. STAT1 interacts with TA-p73 in vitro

To investigate whether STAT1 interacts with TA-p73 we

transfected STAT1 deficient Mouse Embryonic Fibroblast

cells (STAT1�/� MEF) cells with STAT1-a (pSTAT1-a/

MYC) or STAT3 (STAT3/pCDNA3.1+) encoding plasmids

and conducted co-precipitation experiments with GST-TA-

p73 Sepharose. Upon the analysis of GST-TA-p73 Sepharose

complexes by Western blotting using an anti-STAT1 or

-STAT3 antibody (Fig. 1a), STAT1 alone was observed to di-

rectly interact with GST-TA-p73.

To determine which region of the TA-p73 protein sequence

[27] is responsible for interacting with STAT1-a, we next per-

formed co-precipitation experiments with GST-fused deletion

derivatives of TA-p73 (Fig. 1b). PCDNA3.1+ transfected cells

and STAT1-a/MYC expressing cells were used as negative and

positive controls, respectively. As shown in Fig. 1c, GST-TA-

p73 could interact with STAT1-a/MYC within the regions that

mapped to the amino- and carboxyl-terminals (albeit with dif-

fering affinities) with TA-p73 DNA binding domain (DBD)

interacting the strongest. GST-TA-p73 amino acids 313–486

(OD domain) did not exhibit any binding to STAT1-a/MYC.

As a positive control for this experiment, we could demon-

strate the interaction of expressed STAT1-a with GST-

STAT1-a, presumably in the absence of STAT1Y701

phosphorylation as shown previously [28,29]. Additionally,

TA-p73 binding sites for GST-STAT1 were also determined

using co-precipitation. Cells transfected with empty expression

plasmid (pCDNA.1+) or p53-HA expression plasmid were

used as negative and positive controls, respectively. HA-tagged

TA-p73 derivatives and the DeltaN-p73 isoform lacking the N-

terminal transactivation domain [35,37] were expressed in

COS-7 cells and cell supernatants incubated with GST-STAT1

Sepharose. Upon analysis of the co-precipitation complexes,

all TA-p73 derived protein constructs were found to interact

with GST-STAT1 except the construct that encoded p73 ami-

no acids 313–486 (OD domain, Fig. 1d).

Fig. 1. STAT1 directly interacts with TA-p73. (a) STAT1�/�MEF cells were transfected with 1 lg of plasmid encoding STAT1-a/MYC or STAT3.Forty-eight hours after transfection cells were lysed, cleared and the whole cell lysates (WCL) incubated with GST- or GST-TA-p73 Sepharoseovernight. Co-precipitation mixtures were washed, resolved by SDS–PAGE and analysed by Western blotting (WB) using an anti-STAT1 antibody(left panel) and an anti-STAT3 antibody (right panel). (b) Diagramatic representation of the TA-p73-GST fusion proteins used to map the STAT1specific binding site. The Amino-Terminal Domain (NTD) harboring the Transactivation Domain (TA, aa 1-54), DNA Binding Domain (DBD, aa131-310), Oligomerisation Domain (OD, aa 345-380) and the Carboxyl-Terminal Domain (CTD) harboring the Sterile Alpha Motif domain (SAM,aa 484–549) are shown. (c) STAT1�/�MEF cells were transfected with pCDNA3.1+ (1 lg) or STAT1-a/MYC and 48 h after transfection, cells werelysed and cleared whole cell lysates (WCL) were incubated with 5 lg of GST-Sepharose, GST-TA-p73 derived Sepharose conjugates or GST-STAT1a Sepharose conjugates (as revealed by Coomassie staining). Eighteen hours later, co-precipitation mixtures were washed and resolved usingSDS–PAGE and analysed by Western blotting (WB) with an anti-MYC antibody. (d) STAT1�/� MEF cells were transfected with 1 lg ofpCDNA3.1+, p53-HA and the TA-p73-HA derived expression plasmids. Forty-eight hours later, cells were washed, lysed and cleared whole celllysates (WCL) were incubated overnight with approximately 5 lg GST- or GST-STAT1a Sepharose. Co-precipitation mixtures were harvested,washed, resolved and analysed by Western blotting (WB) with an anti-HA antibody (top and lower panels). Equal volumes of WCLs were analysedfor positive expression of the p73-derived proteins (middle panel) and quantities of GST- or GST-STAT1 Sepharose used in the binding assays wererevealed by Coomassie staining.

S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226 1219

Collectively, these data suggest that STAT1-a can directly

interact with TA-p73-HA and that the regions within TA-

p73 that are responsible for this span amino acids 1–312 and

487–637. Similarly, the catalytic subunit of PKA has also been

shown to interact with TA-p73 amino acids 63–130 (TA do-

main) and 469–637 (SAM domain) but not amino acids 311–

483 [30], highlighting that the TA-p73 binding sites for STAT1

are not exclusive and that these sites may also mediate interac-

tions with other signaling proteins.

3.2. STAT1 interacts with TA-p73 in intact cells

As STAT1Y701 and STAT1S727 have been demonstrated

to be key residues in mediating the dimerisation, transloca-

tion of STAT1 and the optimization of STAT1-mediated

transcriptional activity, we next addressed the dispensability

of these residues in mediating the interaction between

STAT1 and TA-p73 in mammalian cells using co-immuno-

precipitation experiments. Therefore, STAT1�/� MEF cells

were co-transfected with pCDNA3.1+, STAT1-a, STAT1-b[31], STAT1Y701F, STAT1S727A and pTA-p73-HA, lysed

and TA-p73 immunoprecipitated using an anti-HA antibody.

Upon analysis of co-immunoprecipitation complexes, TAp73-

HA was found to immunoprecipitate equivalently in all

transfections, all STAT1 derivatives were found to positively

interact with TA-p73, with STAT1S727A and STAT1-b inter-

acting weakly (Fig. 2) as reported previously between

STAT1S727A and the CBP co-activator protein [6,32]. As a

positive control, STAT1-a was found to strongly interact

with p53-HA [7]. By comparison, TAp73-HA binding to

STAT1-a was observed to be relatively low, due possibly to

it interacting relatively weakly to STAT1 since the expressed

levels of p53-HA and TA-p73-HA were observed to be quite

equivalent. Moreover, STAT1 expression levels were ob-

served to be fairly consistent, with the exception of STA-

T1Y701F, which either expressed at relatively higher levels

or possesses greater protein stability.

Fig. 2. STAT1 interacts with TA-p73-HA in intact cells. STAT1�/�

MEF cells were transfected with 0.5 lg of TA-p73-HA and 0.5 lgpCDNA3.1+, p53-HA or the indicated STAT1 derived expressionplasmids. Forty-eight hours after transfection, cells were washed, lysedand TA-p73 immunoprecipitated (IP) using an anti-HA antibody(3 lg) or Normal IgG (NIgG) antibody. Immune complexes werewashed and resolved using SDS–PAGE and analysed by Westernblotting (WB) with an anti-STAT1 antibody. Equal volumes of Wholecell lysates (WCL) were also analysed by Western blotting to detectexpression levels of p5 3-HA, STAT1 constructs and pTA-73-HA.

Fig. 3. STAT1 and TA-p73-HA localisation in STAT1�/� MEF cells.STAT�/� MEF cells grown on cover slips were transfected with 0.5 lgTA-p73-HA (or p53-HA) and 0.5 lg STAT1 derived expressionplasmids (in duplicate). Forty-eight hours after transfection, cells werewashed, fixed using paraformaldehyde, permeablised and stained usingSTAT1-a (anti-MYC, green channel) or anti-STAT1a/b specificantibody (green channel) and anti-HA antibodies (red channel). Coverslips were mounted and visualized using laser scanning confocalmicroscopy. Regions of co-localization are indicated by white arrows.Cells expressing STAT1-a/MYC (green channel) and TA-p73-HA (redchannel) were also stained with the corresponding non-immune IgGantibodies (NIgG) as negative controls.

1220 S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226

3.3. Subcellular localization of STAT1 and TA-p73 in

STAT1�1� MEF cells

Considering that STAT1 derivatives display differential bind-

ing by pTA73 (Fig. 2), in beginning to determine whether this

is due to differing subcellular compartmentalization of these

two proteins, we utilized laser scanning confocal microscopy to

image expressed STAT1 derivatives and pTA73. STAT1�/�

MEF cells were transfected using plasmids that encode

STAT1-a/MYC, STAT1Y701F, STAT1-b, STAT1S727A with

HA-tagged TA-p73 (pTA-p73-HA). STAT1-a/MYC and p53-

HA were utilized as a positive control and observed to be

respectively present in the cytoplasm and nucleus as seen pre-

viously [7]. In Fig. 3, STAT1-a, STAT1-b [31] and STAT1-

S727A were seen to be present in distinct compartments

within the cytoplasm (presumably as latent STAT1) which is

in good agreement with previously published observations

[29,33]. Additionally, all STAT1 derivatives were observed to

show some co-localisation with TA-p73 to varying degrees

with TA-p73-HA within the cytoplasm [34] or nucleus [35].

Collectively, these data suggest that expressed STAT1-a/

MYC and TA-p73-HA can co-localize within the cytoplasm

and nucleus of STAT1�/� MEF cells confirming the specific

interaction of TA-p73 with STAT1 (Fig. 2).

S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226 1221

3.4. Phospho-Y701STAT1 can interact with TA-p73 in vivo

To test the above findings in a cell system that does not uti-

lize the artificial expression of TA-p73 or STAT1, we next con-

ducted co-precipitation experiments between STAT1-a and

GST-TA-p73 using cell supernatants derived from primary

neonatal cardiomyocyte cells (CM), as seen between STAT1

and p53 [7]. Primary CM cells were stimulated with IFN-c,

lysed and cleared supernatants incubated with GST-alone or

GST-TA-p73 protein immobilized on glutathione Sepharose.

As shown in Fig. 4, endogenous phospho-STAT1a and b were

found to positively interact with GST-TA-p73 Sepharose in an

IFN-c stimulatory manner as early as 10 min after stimulation

which was maximal after 45–60 min.

3.5. TA-p73 expression activates the Bax promoter

As we observed STAT1+/+ cells to express relatively low

amounts of TA-p73 (S.M. Soond, unpublished), we first

sought to establish what quantities of expressed pTA73-HA

could activate the Bax promoter in the presence and absence

of IFN-c stimulation. Therefore, STAT1+/+ MEF cells were

transfected with an increasing dose of TA-p73-HA expression

plasmid, followed by IFN-c stimulation before cells were lysed

and fixed volumes of supernatant evaluated for luciferase

activity. As shown in Fig. 5a, we observed a TA-p73 dose

dependent increase in Bax promoter activity (over

pCDNA3.1+ transfected cells), which peaked at 1 lg of trans-

fected TA-p73 DNA. Moreover, upon stimulation of cells with

IFN-c, the relative amounts of luciferase activity was observed

to be reduced by approximately 30%.

3.6. STAT1 negatively regulates TA-p73-mediated Bax

promoter activity

Next we addressed the regulatory effects of STAT1 expres-

sion on TA-p73 mediated Bax promoter activation. Therefore,

Fig. 4. Activated STAT1 interacts with TA-p73 in neonatal primarycardiomyocyte cells. Primary CM (106) were stimulated using 25 ng/mlrat IFN-c for the indicated times. At each time point cells werewashed, lysed and the whole cell lysates (WCL) incubated withapproximately 5 lg GST- or GST-TA-p73 Sepharose overnight. Co-precipitates were washed and resolved using SDS–PAGE followed byWestern blotting (WB) with an anti-phospho-701 STAT1 (p-STAT1)antibody. Quantities of incubated GST- or GST-TA-p73 Sepharosewere revealed by Coomassie staining.

TA-p73-HA plasmid with increasing amounts of pSTAT1-a/

MYC were used to transfect STAT1�/� MEF, cells stimulated

with IFN-c and cleared lysates assayed for luciferase reporter

activity. Fig. 5b shows that with increasing amounts of

STAT1-a expression, decreasing levels of Bax promoter

activity were observed. Additionally, when 1 lg of STAT1-aencoding plasmid was used in the transfection mix followed

by IFN-c stimulation, we observed �50% reduction in Bax

promoter activity. In this manner, STAT1-a expression in

the absence of TA-p73-HA expression, followed by IFN-cstimulation had no significant change on Bax promoter activ-

ity in comparison to pCDNA3.1+ transfected cells, eliminating

the possibility of STAT1-a modulating the Bax promoter di-

rectly in our system (S.M. Soond, unpublished). Collectively,

these data suggest that STAT1-a downregulates TA-p73 med-

iated Bax promoter activity and that this is further suppressed

in the presence of IFN-c stimulation in our reconstituted

STAT1�/�MEF cell based system, which is consistent with

the observations see in STAT1+/+ cells expressing TA-

p73-HA (Fig. 5a).

3.7. STAT1 Ser-727 is critical in regulating TA-p73 mediated

Bax expression

In view of maximum STAT activation requiring the phos-

phorylation of Tyr-701 by JAK kinase and the possible

MAPK phosphorylation of STAT1 Ser-727 [4,5], we next

sought to address the dispensability of these residues in

STAT-a mediated abrogation of TA-p73 induced Bax pro-

moter activation. Therefore, we transfected STAT1�/� MEF

cells with pCDNA3.1+, pTA-p73-HA and STAT1-a/MYC,

pSTAT1-b, pSTAT1Y701F or pSTAT1S727A expression

plasmids , stimulated them for 24 h using IFN-c before cell

lysates were prepared and assayed for Bax promoter activity.

As seen from Fig. 5c, plasmids encoding STAT1-a, STAT1-band STAT1Y701F resulted in reduced Bax promoter activa-

tion in the presence and absence of IFN-c stimulation. How-

ever, STAT1S727A was observed to show elevated levels of

Bax promoter activation in the absence and presence of

IFN-c stimulation, in comparison to STAT1-a. Collectively,

these data suggest that under IFN-c stimulatory and non-

stimulatory conditions, abrogation of TA-p73 mediated Bax

promoter activation is heavily dependent upon modification

of STAT1 Ser-727 in STAT1�/� MEF cells. Considering

that STAT1-b and STAT1S727A lack the Ser-727 phosphor-

ylation site and yet yield obvious differences in p73 mediated

Bax promoter regulation, we next sought to address whether

this may be due to changes in STAT1 or TA-p73 sub-cellu-

lar location upon IFN-c stimulation of cells. Therefore,

STAT1�/� cells transfected with expression plasmids encoding

STAT1-a/MYC, STAT1-b and STAT1S727A were stimulated

with mouse IFN-c and the cells fixed, permeablised and

stained for STAT1 and TA-p73-HA expression. Cells were

imaged using laser scanning confocal microscopy (Fig. 6)

and STAT1 found to localise to the nucleus (as did STA-

T1727A), whereas STAT1-b was seen to localise to a perinu-

clear compartment mainly composed of vessicular structures,

implying that TA-p73 may (at least in part) be spatially reg-

ulated by STAT1-a and STAT1-b. Moreover, STAT1Ser-

727 and the surrounding protein sequence may be of relative

importance in accounting for the relative differences seen in

IFN-c mediated TA-p73 Bax promoter regulation upon

STAT1 expression.

Fig. 5. Bax promoter activation is abrogated in STAT1+/+MEF cells expressing STAT1-a for which STAT1 Ser-727 is required. (a) STAT1+/+ cellswere transfected with pCDNA3.1+ or with the stated amounts of TA-p73-HA expression plasmid along with 250 ng Bax-luciferase and 50 ng b-galactosidase/pRSV. Transfecting DNA was kept constant using pCDNA3.1+ plasmid. Forty-eight hours after transfection, cells were either left un-stimulated (white bars) or stimulated with mouse IFN-c (50 ng/ml, black bars) for 24 h. Cells were washed and lysed before fixed volumes of lyatewere analysed for relative luciferase activity. Luciferase activity was standardized for transfection efficiency, and is expressed as fold activation overbasal. Whole cell lysates were also analysed for TA-p73 (TA-p73-HA), activated phospho-Y701STAT1 (pSTAT1), and GAP DH expression usingWestern blotting. (b) STAT1�/� cells were transfected with 1 lg of pCDNA3.1+ or TA-p73-HA expression plasmid along with the stated amounts ofSTAT1-a/MYC expression plasmid and 250 ng Bax-luciferase and 50 ng b-galactosidase/pRSV. Transfection DNA was kept constant usingpCDNA3.1+ plasmid. Forty-eight hours after transfection, cells were either left un-stimulated (white bars) or stimulated with mouse IFN-c (50 ng/ml, black bars) for 24 h. Cells were washed and lysed before fixed volumes of lysate were analysed for relative luciferase activity. Luciferase activitywas standardized for transfection efficiency, and is expressed as fold activation over basal. Whole cell lysates were also analysed for TA-p73 (TA-p73-HA), STAT1-a, activated phospho-Y701STAT1 (pSTAT1), and GAP DH expression using Western blotting. (c) STAT1�/� cells were transfectedwith 1 lg of pCDNA3.1+ or TA-p73-HA expression plasmid along with 2 lg STAT1 expression plasmids and 250 ng Bax-luciferase and 50 ng b-galactosidase/pRSV. Transfection DNA was kept constant using pCDNA3.1+ plasmid. Forty-eight hours after transfection, cells were either left un-stimulated (white bars) or stimulated with mouse IFN-c (50 ng/ml, black bars) for 24 h. Cells were washed and lysed before fixed volumes of lyatewere analysed for relative luciferase activity. Luciferase activity was standardized for transfection efficiency, and is expressed as fold activation overbasal. Whole cell lysates were also analysed for TA-p73 (TA-p73-HA), STAT1-a (STAT1), activated phospho-Y701STAT1 (pSTAT1), and GAPDH expression using Western blotting.

1222 S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226

3.8. STAT1 negatively regulates Bax protein expression

To extend our observations from above, we next analysed the

expression of the Bax protein under IFN-c stimulatory condi-

tions in STAT1+/+ and STAT1�/� MEF cells. Therefore, equal

quantities of MEF cells were seeded and left un-stimulated or

stimulated for 1–24 h using IFN-c and analysed by Western

blotting using a Bax specific antibody. From the data presented

(Fig. 7), STAT1+/+ MEF cells expressed less Bax protein in

comparison to STAT1�/� cells, which was observed to signifi-

cantly decrease in STAT1+/+ cells after 24 h of IFN-c stimula-

tion in comparison to STAT1�/� cells, in cell lysates equivalent

in b-actin expression. Collectively, these observations suggest

that IFN-c stimulation of MEF cells up to 24 h, results in

downregulation of Bax protein expression and that this occurs

in a STAT1 expression-dependent manner.

3.9. STAT1 downregulates TA-p73 mediated Bax promoter

activation independently of p53

In light of the observation that p53 may positively regulate

the expression of the TA-p73 inhibitor DeltaN.p73 [36], we

Fig. 6. Sucellular location of STAT1 and TA-p73 in IFN-c stimulatedcells. STAT1�/� MEF cells (seeded on coverslips) were transfectedwith 0.5 lg of TA-p73-HA along with 0.5 lg of the indicated STAT1expression plasmids. Forty-eight hours after transfection, cells werestimulated for 24 h with mouse IFN-c (50 ng/ml), fixed, permeablised,stained for STAT1 (green channel), TA-p73-HA (red channel) and thenucleus (blue channel, using DAPI) and imaged using laser scanningconfocal microscopy. Regions of co-localisation are indicated witharrows.

Fig. 7. STAT1 expression downregulates Bax protein expression fol-lowing IFN-c stimulation. STAT1�/� and STAT1+/+ MEF cells(0.5 · 105) were left un-stimulated or stimulated for the indicatedhours (hrs) using 50 ng/ml mouse IFN-c. Cells were washed, lysed andproteins resolved using SDS–PAGE and analysed by Western blottingwith anti-Bax (-Bax), anti-phosphoY701-STAT1 (-pSTAT) and anti-bactin (-Actin) antibodies.

S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226 1223

next addressed whether IFN-c mediated STAT1-dependent

downregulation of Bax promoter activity by TA-p73 occurs

in a p53 dependent manner. To this end, we generated

STAT1/p53�/� ‘double-knockout’ MEF cells, which we co-

transfected with the Bax-luciferase reporter construct, TA-

p73-HA expression plasmid with an increasing dose of

STAT1-a expression. Forty-eight hours after transfection, cells

were stimulated with IFN-c for 24 h after which, cell lysates

were assayed for relative luciferase activity. As shown in

Fig. 8a, STAT1 expression in these cells resulted in a dose-

dependent decrease in Bax reporter activity, which could be

inducibly downregulated in the presence of IFN-c stimulation.

Additionally, we attempted to detect endogenous Bax

protein expression by comparing p53�/� cells and STAT1/

p53�/� cells during a time course of IFN-c stimulation (as in

Fig. 7). However, our attempts to detect the Bax protein in this

analysis were unsuccessful (S. M. Soond, unpublished) and we

propose that this could be due to Bax protein expression hav-

ing an intrinsic requirement for p53 expression, as reported

previously [37,38].

3.10. STAT1 Tyr-701 and Ser-727 residues are both important

in TA-p73 mediated Bax promoter downregulation in the

absence of p53 expression

Next asked how dispensable residues STAT1Y701 or

STAT1S727 are in regulating the transcriptional activity of

TA-p73 independently of p53. Therefore, STAT1/p53�/�

MEF cells were transfected with pCDNA3.1+ alone, pTA-

p73-HA and pCDNA3.1+, pSTAT1-a/MYC, pSTAT1Y701A

or pSTAT1S727A expression plasmid. Forty-eight hours later

cells were stimulated with IFN-c for 24 h before cell lysates

were prepared and assayed for relative luciferase activity. As

shown in Fig. 8b, STAT1-a expression followed by IFN-cstimulation resulted in downregulation of TA-p73 mediated

Bax promoter activity which was significantly reduced upon

expression of STAT1Y701F and STAT1S727A with or with-

out IFN-c stimulation. These data suggest that for the

STAT1-a dependent downregulation of TA-p73 mediated

Bax promoter activity, STAT1-a requires residues Tyr-701

and Ser-727. This is in contrast to when STAT1�/p53+ MEF

cells are used in such an experiment (Fig. 5c), where STAT1

Ser-727 was seen to be important for STAT1 mediated Bax

promoter abrogation.

4. Discussion

Based on the knowledge that TA-p73 and p53 can bind iden-

tical transcriptional regulatory sequences, it is no surprise that

both of these transcription factors mediate the regulation of an

overlapping repertoire of genes, such as p21, PUMA, p53R2

and Bax. In view of this, one of the key challenges in this field

is how these transcription factors may be biochemically regu-

lated, giving rise to differential and specific down-stream gene

Fig. 8. STAT1 expression in STAT1/p53�/� MEF cells abrogates TA-p73 mediated Bax promoter activity for which STAT1 Tyr-701 andSTAT1 Ser-727 are required. (a) STAT1/p53�/� MEF cells weretransfected with 1 lg of pCDNA3.1+ or TA-p73-HA expressionplasmid with the shown quantities of STAT1-a expression plasmids,250 ng Bax-luciferase and 50 ng b-galactosidase/pRSV plasmids. DNAamounts were kept constant using pCDNA3.1+ plasmid. Forty-eighthours after transfection, cells were either left un-stimulated (whitebars) or stimulated with mouse IFN-c (50 ng/ml, black bars) for 24 h.Cells were washed and lysed before fixed volumes of lysate wereanalysed for relative luciferase activity. Luciferase activity wasstandardized for transfection efficiency, and is expressed as foldactivation over basal. Whole cell lysates were also analysed for TA-p73(TA-p73-HA), STAT1-a (STAT1), activated phospho-Y701STAT1(pSTAT1), and GAP DH expression using Western blotting. (b)STAT1/p53�/� MEF cells were transfected with 1 lg of pCDNA3.1+or TA-p73-HA expression plasmid along with 0.5 lg STAT1 expres-sion plasmids, 250 ng Bax-luciferase and 50 ng b-galactosidase/pRSV.DNA amounts were kept constant using pCDNA3.1+ plasmid. Forty-eight hours after transfection, cells were either left un-stimulated (whitebars) or stimulated with mouse IFN-c (50 ng/ml, black bars) for 24 h.Cells were washed and lysed before fixed volumes of lysate wereanalysed for relative luciferase activity. Luciferase activity wasstandardized for transfection efficiency, and is expressed as foldactivation over basal. Whole cell lysates were also analysed for TA-p73-HA (TAp73-HA), STAT1-a (STAT1), activated phospho-Y701STAT1 (pSTAT1), and GAP DH expression using Westernblotting.

1224 S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226

regulatory effects. Whereas results published from this labora-

tory have addressed this in light of p53 gene regulation by acti-

vated STAT1-a in the presence of DNA damaging agents and

ischaemia-reperfusion injury [7,20], the regulatory role played

by STAT1 on TA-p73 directed Bax expression during IFN-csignalling, remains poorly understood.

To date, the Bax promoter has been demonstrated to be reg-

ulated by a number of proteins which include p53 and TA-p73.

Data published previously have demonstrated that the Bax

promoter can be activated upon TA-p73 overexpression

[30,39,40]. Moreover, overexpression of p73 has been reported

in various tumour types when compared with normal tissues

[41–45] and has been reported to confer resistance to p53-med-

iated apoptosis induced by certain chemotherapeutic agents

[46]. This is demonstrated in p73-deficient neurons exhibiting

enhanced apoptosis in vivo [47]. Using an approach that uti-

lized TA-p73 expression, we demonstrated that STAT1 expres-

sion downregulated Bax promoter activity in an IFN-cdependent manner and Ser-727 phosphorylation is critical

for this to occur in a p53-dependent manner within

STAT1�p53+MEF cells. Although, maximum activation of

STAT1 is dependent upon the phosphorylation of Y701 by

JAK kinase [5], STAT1Ser-727 phosphorylation has been

shown to be critical for maximum STAT1 activation and

STAT1-mediated cell death [4,7,48]. However, STAT1-b [31]

which lacks the STAT1Ser-727 phosphorylation site and car-

boxyl-terminal amino acids (which are otherwise present in

STAT1-a) also produces diminished Bax promoter activity in

contrast to STAT1S727A (in STAT1�/� MEF cells, Fig. 5c),

in addition to it being able to interact with TA-p73 (Figs. 4a

and 2) and co-localise with it (Fig. 6) suggesting that the

underlying mechanism is clearly very complex (which could

be STAT1-a or STAT1-b specific) and that TA-p73/STAT1

protein trafficking may be of importance in regulating TA-

p73 mediated Bax expression in a p53 dependent manner.

Moreover, in the absence of p53 expression, we observed that

STAT1 required residues Tyr-701 and Ser-727 to downregulate

TA-p73 mediated Bax promoter activation. We propose that

this may highlight a distinct mechanism (however complex)

by which TA-p73 (in comparison to p53) may regulate Bax

promoter regulation based upon the phosphorylation status

of STAT1 and the presence of p53.

In a manner similar to p53, TA-p73 is stabilized at the pro-

tein level in response to DNA damaging agents permitting it to

mediate its pro-apoptotic transcriptional activity [49]. Positive

stabilizers of TA-p73 protein and activity include MDM2 [50]

– as in the case of p53 [51–53], YAP [54], and phosphorylation

by PKC-d [55]. Conversely, TA-p73 activity can be negatively

regulated by cyclin G [56], SUMO-1 [57], PIAS-1 [19] calpain I

[58], UFD2a [39], PKA-Cb [30] and Itch [59]. Other identified

binding partners to TA-p73 include HMGB1 and HMGB2,

which stimulate or abrogate the binding of TA-p73 to p53

responsive elements (within the Bax promoter) in a cell specific

manner [40]. With these observations in mind, how STAT1-aregulates the activity of endogenous TA-p73 induced Bax pro-

moter and protein expression, mechanistically, requires further

study. From Western blotting, analysis of the whole cell lysates

for TA-p73 expression (Fig. 5), we do not observe a decrease in

the stability of TA-p73 in response to STAT1 expression and

IFN-c stimulation that may explain the observed IFN-c med-

iated inducible reduction in Bax promoter activity. Although,

this may be a consequence of artificially expressing TA-p73,

the observation that Bax promoter activity is marginally in-

creased in response to IFN-c stimulation in pCDNA3.1+

transfected STAT1+/+ MEF cells (Fig. 5a) argues against this

possibility. Additionally, based upon the observation that

STAT1 can interact strongly with the DBD domain of TA-

p73 (Fig. 1b), this may suggest that STAT1 may interact with

TA-p73 thereby reducing the ability of TA-p73 to bind DNA

and activate transcription.

S.M. Soond et al. / FEBS Letters 581 (2007) 1217–1226 1225

The consequential modulation of TA-p73 by activated

STAT1 may also be mediated indirectly through the induction

of Interferon-Regulatory Factor-1 [60] and remains to be ex-

plored further. However, evidence linking IFN-c stimulation

with the downregulation of the Bax gene has been reported

previously in MDA-MB213 breast carcinoma [61] and rat liver

cells [62], supporting our observations and highlighting an

important role for STAT1 as an upstream negative-regulator

of the Bax promoter via TA-p73.

In summary, our findings suggest that STAT1 can physically

associate with TAp73 and that in MEF cells can downregulate

TAp73 mediated Bax promoter activation and Bax protein

expression. Our observations also suggest that this decrease

in Bax promoter activity is induced upon IFN-c stimulation

of MEF cells and that STAT1Ser-727 modification is required

for this. In consideration of previously published data, where

STAT1 has been shown to interact and potentiate the activity

of p53 in response to DNA damage and cellular stress [7], we

propose that STAT1-dependent reduction of TAp73 mediated

Bax gene regulation may present a potential mechanism that

allows selective regulation of the TAp73 and TAp53 family

of transcription factors for a common set of transcriptionally

regulated genes.

Acknowledgments: We acknowledge Mr. Sean Barry, Drs. Kevin Law-rence and James McCormick for their intellectual input and Ms. DalyaSoond for proofreading this manuscript. We also express our gratitudeto the British Heart Foundation who funded this study.

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